Parkinson's disease is affecting people from 30 years of age and older. Mean age at onset is approx. 60 years. The major clinical symptoms are rigidity, bradykinesia and resting tremor. In addition the disease can show a range of other symptoms such as hypotension, cognitive impairment, postural instability, and many more.
The disease is primarily a basal ganglia disorder caused by degeneration of the nigrostriatal dopaminergic system in the brain (nerve cells using dopamine (DA) as their signaling substance, located in the substantia nigra of the brain stem projecting to the putamen and caudate nucleus). The disorder is progressive over many years.
The current treatment standard is based on substitution of dopamine by addition of L-dopa (which is converted to dopamine in the brain), or other dopamine-receptor stimulating agents. Although current treatment strategies aimed at substitution of the dopamine deficiency are often very efficient in the early phase of the disease (up to 7-10 years), eventually most patients start to experience diminishing treatment response and increasing adverse events. The most problematic of these is the L-dopa-induced dyskinesias that appear as a result of treatment with the current drug-of-choice, L-dopa, or dopamine agonists. Since patients with Parkinson's disease tend to live longer and longer with their disease, due to improved treatments in recent years, the L-dopa induced dyskinesia poses an increasing problem especially for patients with early onset of the disease. There are today few treatment options for dyskinesias, and these are often complicated and the access is limited.
One approach that has been tested in preclinical animal models of Parkinson's disease is to refine the classical pharmacological dopamine replacement strategy by using a gene therapy approach to obtain a local dopamine replacement in the putamen and caudate nucleus where the dopamine deficiency is most advanced. This approach is called the “enzyme replacement strategy”. The rationale for this treatment stems from clinical observations in Parkinson's disease (PD) patients, which suggested that severe abnormal involuntary movements (i.e., dyskinesias), induced by oral L-DOPA medication, could be alleviated by L-DOPA or DA agonists infused either via the intravenous or duodenal route. Thus, the current hypothesis is that dyskinesias develop, at least in part, due to the intermittent, pulsatile supply of DA that the oral L-DOPA delivery paradigm gives rise to. These patients benefit from continuous DA stimulation also by dramatic reduction in total time spent in “off” state.
Three different enzymes are necessary for the production of dopamine, namely tyrosine hydroxylase (TH), GTP-cyclohydrolase 1 (GCH1) and aromatic amino acid decarboxylase (AADC). The two first regulate the production of L-DOPA from tyrosine (a dietary amino acid) while AADC converts L-DOPA to dopamine. None of these enzymes are unique to dopaminergic neurons but may also be present in non-dopaminergic cells. The addition of these enzymes to the denervated target area can result in production of L-DOPA or dopamine locally. The advantage of this strategy may be that it provides a constant production of L-DOPA in relative to the conventional oral therapies where the L-DOPA plasma levels (and also brain levels) are fluctuating. It also localizes the treatment to the brain area in need for substitution while other parts of the body are not “treated” resulting in a favourable effect versus side effect-ratio.
Published preclinical data using this approach have provided the following observations:                1. Expression of all three genes can be obtained in the putamen and caudate nucleus by transduction using multiple rAAV vectors [Kaplitt M G, et al: Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain; Nat Genet. 1994 8 148-54; Mandel R J, et al: Characterization of intrastriatal recombinant adeno-associated virus-mediated gene transfer of human tyrosine hydroxylase and human GTP-cyclohydrolase I in a rat model of Parkinson's disease; J Neurosci 1998 18 4271-84; Shen Y, et al: Triple transduction with adeno-associated virus vectors expressing tyrosine hydroxylase, aromatic-L-amino-acid decarboxylase, and GTP cyclohydrolase I for gene therapy of Parkinson's disease; Hum Gene Ther 2000 11 1509-19].        2. The efficiency of TH is dependent on GCH1 (which produces the co-factor tetrahydrobiopterin, BH4). Mandel and collaborators have shown this by measuring levels of L-dopa using micro dialysis [Mandel R J, et al: Characterization of intrastriatal recombinant adeno-associated virus-mediated gene transfer of human tyrosine hydroxylase and human GTP-cyclohydrolase I in a rat model of Parkinson's disease; J Neurosci 1998 18 4271-84].        3. In a monkey model of Parkinson's disease (the MPTP-model) expression of AADC can result in more efficient conversion of oral L-dopa and through this mechanism improve function in a monkey UPDRS motor score (UPDRS is the standard clinical evaluation scale for Parkinson symptoms) [Bankiewicz K S, et al: Long-term clinical improvement in MPTP-lesioned primates after gene therapy with AAV-hAADC; Mol Ther 2006 14 564-70].        4. Expression of all three genes can result in improved function in both rat models [Shen Y, et al: Triple transduction with adeno-associated virus vectors expressing tyrosine hydroxylase, aromatic-L-amino-acid decarboxylase, and GTP cyclohydrolase I for gene therapy of Parkinson's disease; Hum Gene Ther 2000 11 1509-19] and monkey models [Muramatsu S, et al: Behavioral recovery in a primate model of Parkinson's disease by triple transduction of striatal cells with adeno-associated viral vectors expressing dopamine-synthesizing enzymes. Hum Gene Ther 2002 13 345-54] of Parkinson's disease].        5. Expression of TH and GCH1 is sufficient to obtain striatal L-dopa levels that can result in functional improvement in a rat model of Parkinson's disease and can furthermore significantly reduce the L-dopa induced dyskinesia [Kirik D, et al: Reversal of motor impairments in parkinsonian rats by continuous intrastriatal delivery of L-dopa using rAAV-mediated gene transfer; Proc Natl Acad Sci 2002 99 4708-13; Carlsson et al: Reversal of dyskinesias in an animal model of Parkinson's disease by continuous L-DOPA delivery using rAAV vectors; Brain 2005 128 559-69]. However, these studies were conducted using two separate AAV serotype 2 vectors each containing either the GCH1 gene or the TH gene, both under the control of a large synthetic promoter (chicken b-actin promoter containing a rabbit gamma-globulin intron, preceded with an enhancer element from the cytomegalovirus promoter, termed as the chicken b-actin, CBA, promoter). As such, there was no possible way to control the expression ratio of the two genes at a cellular level; nor did the promoter enable expression limited to neurons.        
In respect of the current state of the art within the field of the present invention, Sun et al (2004), describes a non-AAV viral expression vector with two transcriptional units, each regulated by a neuron-specific promoter. It does not describe the relative level of transcription of the two units. In vitro there is comparable amounts of cells expressing TH and GCH-1 when transduced with both the 3-gene and the 4-gene vector. In both cases TH and GCH-1 are on different transcripts. In the 4-gene vector GCH-1 is translated from an IRES site (after the VMAT-2 ORF).
Shen et al 2000 describes co-transduction of HEK-293 cells with AAV-TH, AAV-AADC and AAV-GCH-1. In a titration study, 293 cells were transduced with AAV-TH and AAV-AADC and varying amounts of AAV-GCH-1. The results show an increase in both L-dopa and dopamine with increasing titer of AAV-GCH-1. AAVGCH-1 was tested in titers up to the same as for AAV-TH. The described ratios are 1:10, 1:2 and 1:1 (AAV-GCH-1:AAV-TH). In vivo gene therapy was conducted with a 1:1 ratio of the two vectors (with and without AAV-AADC).
Kirk et al, 2002 and Carlsson et al 2005 describe co-administration of a 1:1 mix of AAV-TH and AAVGCH1, in which the titer of AAV-TH is approximately 3.5 times that of AAV-GCH-1 (ratio of 1:3.5). Neither of these references states why this ratio was used.
U.S. Pat. No. 7,419,829 (Oxford Biomedica) describes a mutated WPRE element and its use in a three-gene vector (EIAV) with TH, AADC, and GCH-1 separated by IRES sequences. The WPRE element with enhance the expression of the three genes to the same extent.
WO 96/05319 (Arch Development) describes dicistronic vectors with either an IRES site or the promoter in the 5′ retroviral LTR, which controls expression of both an upstream and a downstream cistron. In a double transduction experiment with fibroblasts, they disclose a TH activity of 242.6 pmol/mg/min and a GCH1 activity of 35.8 pmol/mg/min. This translates into a ratio of 1:6.8 between the two enzymes on an activity basis. In addition the reference describes an optimum BH4 concentration (500 QM) in order to achieve maximum TH activity. L-DOPA concentration in TH-transduced cells was maximum beyond 50 QM BH4 and did not increase further with higher concentrations of BH4.
None of the mentioned references describe an AAV vector with a construct coding for both TH and GCH-1. All the one-vector systems in the prior art coding for both TH and GCH-1 have been made in viral vectors that include much larger pieces of nucleic acid. Most of the one-vector systems of the prior art additionally comprise an expression construct coding for AADC. AAV vectors present advantages over the one vector systems based on HSV, EIAV and Retrovirus for clinical purposes. In addition, the absence of AADC is also an advantage over the prior art since this leads to generation of L-DOPA in the transduced cells instead of DA.